The invention relates to a novel anticoagulant substance obtained from human urine, a process for its preparation and a pharmaceutical composition comprising the said substance for prevention and/or treatment of diseases related to the disorders in blood coagulation system.
As anticoagulant agents, heparin and antithrombin III are presently in use. Thrombolytic agents in use include urokinase, which is obtained from human urine or from cultured kidney cells, and streptokinase, which is extracted from beta-hemolytic streptococci. In addition, tissue plasminogen activator is now being developed.
On the other hand, it is well known that these drugs have side effects such as tendency of bleeding, and their anticoagulant or thrombolytic effect are not sufficient for the clinical use. In the field of fundamental investigation, an unknown substance was recently purified from rabbit lung extract and was identified as a novel physiological anticoagulant. The substance was named thrombomodulin [N. L. Esmon et al., J. Biol. Chem., Vol. 257, p.859 (1982)]. Thrombomodulin has two modes of action; an anticoagulant activity based on its anti-thrombin effect, and a fibrinolytic effect based on its stimulatory effect on thrombin-catalyzed protein C activation. Thrombomodulin is a receptor of thrombin on the endothelial cell surface, and, by binding thrombin, directly inhibits its procoagulant activity. Moreover, thrombin-thrombomodulin complex activates protein C which possesses a potent anticoagulant effect and a thrombolytic effect [I. Maruyama et al., J. Clin. Invest., Vol. 75, p.987 (1985)]. Since thrombomodulin exhibits not only an anticoagulant activity but also an enhancing effect on thrombolytic system, it is expected to be very useful for the treatment of blood coagulation disorders.
Since the thrombomodulin molecule mainly consists of peptides, thrombomodulin derived from the human, which has little antigenicity, ought to be administered to patients in order to avoid side effects such as anaphylactic shock. In regard to the isolation of human-derived thrombomodulins, there are some reports as described below. In following explanation, molecular weights of the human-derived thrombomodulins, if not otherwise, are the results of measurements by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under non-reduced condition.
P. W. Majerus et al. purified thrombomodulin from human placenta and reported its molecular weight as 75,000 [J. Biol Chem., Vol 259, p.12246 (1984)]. I. Maruyama et al. isolated thrombomodulin from human lung, which has activities similar to those of thrombomodulin from human placenta [J. Clin. Invest. Vol. 75, p.987 (1985)]. N. Aoki et al. isolated thrombomodulin from human placenta and reported its molecular weight as 71,000 [Thrombosis Res., Vol. 37, p.353 (1985); and Japanese Patent Application Laid-Open Specification No. Sho 60-199819]. K. Suzuki et al. partially purified thrombomodulin from human platelets and determined its molecular weight as 78,000. They concluded in the report that all of the thrombomodulins obtained from human platelets, ones from human placenta and ones from human lung hemangio endothelial cells have similar activities by comparing their behaviors in SDS-PAGE, affinities to thrombin and substrate-affinities to protein C [J. Biochem., Vol. 104, p.628 (1988)].
There are also some reports about other substances which have activities similar to above-mentioned human thrombomodulins as described below.
P. W. Majerus et al. partially purified substances with molecular weights of 63,000 and 54,000 from human serum and also indicated that similar substances exist in human urine [J. Clin. Invest., Vol. 75, p.2178 (1985)]. H. Ishii et al. reported that substances with molecular weights of 105,000, 63,000, 60,000, 33,000, 31,000 and 28,000 (whether these were measured under reduced or non-reduced condition is not clearly described) are excreted in human urine [108th. Yakugakkai abstract 6F05, 11-1 (1988)].
Further, other substances with molecular weights of 200,000, 48,000 and 40,000 from urine [Japanese Patent Application Laid-Open Specification No. Sho 63-30423], and substances with molecular weights of 39,000 and 31,000 [Japanese Patent Application Laid-Open Specification Sho No. 63-146898]from urine are reported.
Meanwhile, by means of genetic engineering techniques, K. Suzuki et al. deduced the entire 557-amino acid sequence of human thrombomodulin from human lung cDNA clones [EMBO Journal, Vol 6. p.1891 (1987)]. They produced a series of peptides, which contain repeated Epidermal Growth Factor (EGF)-like structures in human-thrombomodulin, by recombinant DNA techniques, and measured the effect of each peptide on thrombin-catalyzed protein C activation. Based on the obtained result, they concluded that all of the structure from the fourth through sixth EGF-like structures, which corresponds to 345th through 462nd amino acid residues numbered from amino-terminal of human thrombomodulin, is required for the exertion of thrombomodulin-like activity [J. Biol. Chem., Vol. 264, p.10351 (1989) and 12th. International Conference on Thrombosis and Hemostasis program, p.334 (1989)].
The human thrombomodulins which have been already reported are obtained from human placenta, human lung or human platelets. They are not suitable for mass production because these materials could not be supplied in large quantities. Moreover, these thrombomodulins are difficult to handle, because some detergents are necessary in order to solubilize them. Contamination by detergents is unfavorable for clinical use of these thrombomodulins.
On the other hand, previously reported thrombomodulin-like substances have neither high activity of protein C activation no efficient anticoagulant activity per unit protein. Accordingly, a novel thrombomodulin-like substance which is physiologically more active and more valuable in medical use is earnestly expected.
In the conventional processes for the purification of the thrombomodulin-like substances from human urine, some proteinase inhibitors such as aprotinin or bestatin are used to prevent the substances from decomposition. There are, however, other enzymes such as uropepsin which can not be completely inhibited by these proteinase inhibitors, and a complete prevention of the thrombomodulin-like substances from decomposition can not be easily achieved by these processes.
Further, since thrombomodulin is a glycoprotein, genetic engineering techniques could not provide a substance which possesses sugar chains completely equal to those of human thrombomodulin. Differences in sugar chains may cause unfavorable properties such as some side effects. For these reasons, it has been desired to obtain a thrombomodulin-like substance which resembles more closely to a native human thrombomodulin.